- Design primer pair so that the mutation (s) are near the middle of the primer, and the primer has a melting temperature high enough
- Perform 12-16 rounds of PCR on whole vectors
(Include a control sample where you just incubate with the primary one, the other primary displace water.)
- Check the PCR product on gel; if there is no product, lowering the annealing temperature PCR; if there is a smear, increasing the annealing temperature PCR
- Digest with DpnI for 2 hours at 37 degrees C.
- Transform into competent cells, and plate to plate agar.
- Select a colony and grow bacteria in a culture overnight.
- Purify plasmid, check the concentration and insert DNA sequences to ensure that the desired mutation had occurred, and no more random mutations are present.
- Express your protein of interest via transfection in mammalian cells or other methods.